Abstract Interactions between cancer cells and their microenvironment can facilitate tumor growth and progression by altering gene expression and modulating tumor cell behavior such as by enhancing growth, survival, spread or neovascularization. The transfer of bioactive molecules from one cell to another is a mechanism by which tumor cells interact with other cells within their microenvironment. Non-coding (nc) RNA molecules are capable of effecting genomic changes and can modulate gene expression. Thus, intercellular transfer of ncRNA provides a powerful mechanism by which tumor cells can epigenetically modify their environment. The overall objective of this proposal is to understand the molecular mechanisms by which ncRNA are released within extracellular vesicles and their involvement in tumor cell-stromal cell interactions. The studies described are based on our observations of highly selective release of some ncRNA within extracellular vesicles from hepatocellular cancer (HCC) cells and their contribution to activation of cell signaling pathways in recipient cells. Using human HCC or myofibroblastic cell lines, patient-derived HCC, and primary human hepatic stellate cells, we will characterize vesicles and ncRNA that are functionally involved in inter-cellular RNA signaling. These studies will (a) identify extracellular vesicle ncRNA mediators of tumor-stromal interactions and their functional contribution to tumor growth, (b) define identifying characteristics of ncRNA-carrying vesicles and (c) evaluate ESCRT-II dependent mechanisms by which ncRNA are sorted for release within vesicles during tumor-stromal interactions. Using new approaches for the detection of RNA gene expression and inter-cellular transfer with in-vitro and in-vivo tumor-stromal cell co-culture models, these studies will define the mechanistic relationship between regulated release of extracellular vesicles, functional non-coding RNA and tumor cell- stromal cell interactions. Elucidating the essential contribution of inter-cellular RNA signaling to tumor growth will provide the basis for therapeutic strategies to target these interactions and mechanisms for the treatment or prevention of HCC.